Ewing sarcoma family tumors (EFT) are highly malignant bone and soft tissue tumors that primarily affect children, adolescents and young adults. Despite aggressive local control measures and systemic chemotherapy, over a quarter of patients with localized tumors and nearly all patients with metastatic disease will relapse at distant sites following initial clinical remission. Unfortunately, the outlook for these patients is dismal and novel approaches to therapy are desperately needed. One ofthe biggest impediments to improving outcomes and quality of life for patients with EFT is our inability to predict who is at risk for metastatic relapse and to effectively identify and target the mechanisms that underlie this process. The studies outlined in this proposal aim to address these critical gaps in our knowledge. It is our overall goal to improve outcomes for patients with EFT by preventing metastatic relapse. In an effort to achieve this goal we will address three specific aims. First, we will use studies of cell lines to evaluate the role of CXCR4 positive EFT cells in mediating EFT metastasis. We will also determine if invasion downstream of CXCR4 is mediated by and dependent on RhoA. Second, we will test small molecule inhibitors of the RhoA/MKL transcriptional axis in vitro, ex vivo and in vivo to evaluate their efficacy as novel agents forthe prevention of EFT metastasis. Third, we will use retrospectively and prospectively collected EFT samples to validate whether expression of G-protein coupled receptors can be used to predict high-risk disease in newly diagnosed patients. Demonstration that CXCR4, CXCR7 and/or LGR5 expression can be used to identify patients at high risk of metastatic relapse will allow classification of patients into clinical risk categories. In turn, this will allow for treatment stratification and identification of patients who should be included in future trials that are designed to prevent relapse in high-risk patients.